Metabolic enhancement of developmental pausing (TS cells)

Embryonic stem (ES) cells and embryos reversibly pause via chemical mTOR inhibition. In this study, we investigate the tissue-specific response to mTORi-induced pausing in ES and trophoblast stem (TS) cells. To resolve the sequential rewiring of the proteome, we conducted a time-series proteomics experiment at 1, 3, 6, 12, 24, and 48 hours upon induction of pausing, and at 1, 3, 6, 12, 24, and 48 hours upon release of pausing in ES and TS cells. We find that ES, but not TS cells pause reversibly. To optimise developmental pausing conditions, we reasoned that by understanding the difference in pausing response of ES and TS cells, we could identify which pathways are essential for pausing. We found that KEGG pathways related to amino acid degradation, fatty acid degradation, and DNA repair are upregulated in ES cells, but downregulated in TS cells during entry into pausing. Moreover, by targeted metabolomics, we found a depletion of short chain carnitines in the paused ES cells. To extend the length of developmental pausing, we supplemented paused embryos with L-carnitine. The L-carnitine supplementation facilitates lipid usage and prolongs the pausing length by 19 days through the establishment of a more dormant state.胚胎干细胞（Embryonic Stem, ES）与胚胎可通过化学性哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）抑制实现可逆性休眠。本研究旨在探究胚胎干细胞与滋养层干细胞（Trophoblast Stem, TS）对mTOR抑制剂诱导休眠的组织特异性响应。为解析蛋白质组的时序重排过程，我们分别在休眠诱导后的1、3、6、12、24及48小时，以及休眠解除后的1、3、6、12、24及48小时，对两类细胞开展时间序列蛋白质组学实验。研究结果显示，胚胎干细胞可发生可逆性休眠，而滋养层干细胞则无此特性。为优化发育性休眠的培养条件，我们推测：通过解析胚胎干细胞与滋养层干细胞对休眠诱导的响应差异，可明确调控休眠过程的关键通路。本研究发现，在进入休眠阶段时，与氨基酸降解、脂肪酸降解及DNA修复相关的京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路在胚胎干细胞中呈上调表达，而在滋养层干细胞中则呈下调表达。此外，通过靶向代谢组学分析，我们发现处于休眠状态的胚胎干细胞中短链肉碱出现显著耗竭。为延长发育性休眠的持续时长，我们向处于休眠状态的胚胎中添加了L-肉碱。补充L-肉碱可促进脂质代谢利用，并通过构建更为稳定的休眠状态，将休眠时长延长19天。